JP3565727B2 - Method for removing fumarase activity, microorganism obtained by this method, and method for producing optically active aminopolycarboxylic acid using this microorganism - Google Patents

Description

【００１２】
で示される光学活性アミノポリカルボン酸の製造方法であって、上記のフマラーゼ活性が低減された微生物またはその処理物からなる触媒の存在下で、フマル酸と、一般式［２］
【００１３】
【化２】

【００１４】
で示されるアミノ基を有する化合物とを反応させること、および、その結果得られた反応混合物から該光学活性アミノポリカルボン酸を単離することからなり、上記式［１］および式［２］において、Ｒ_１およびＲ_２は、互いに同一でも異なっていてもよく、それぞれ水素原子、置換または無置換のアルキル基、置換または無置換のシクロアルキル基、置換または無置換のアリール基であり、但し、Ｒ_１とＲ_２は共に水素原子であることはなく、該置換基はアミノ基、シアノ基、ニトロ基、ハロゲン、ヒドロキシル基、カルボキシル基およびエーテル基からなる群から選択され；Ｒ_３およびＲ_４はＲ_１とＲ_２と同一であるか、または、Ｒ_１とＲ_２のアミノ基の少なくとも１個がその窒素原子を介してコハク酸のエチレン基の炭素原子と結合した構造を有する基を表わす、前記方法を提供する。
【００１５】
ここで、アミノ基を有する化合物は、例えば、エチレンジアミン、１，３−プロパンジアミン、２−メチル−１，３−プロパンジアミン、２−ヒドロキシ−１，３−プロパンジアミン、１，４−ブタンジアミン、１，５−ペンタンジアミン、１，６−ヘキサンジアミン、１，２−シクロヘキサンジアミン、１，３−シクロヘキサンジアミン、１，４−シクロヘキサンジアミン等の炭素数２〜６のアルカン−またはシクロアルカン−ジアミン類；１，２−フェニレンジアミン、１，３−フェニレンジアミン、１，４−フェニレンジアミン等のフェニレンジアミン類；ならびにグリシン、３−アミノプロピオン酸、２−アミノプロピオン酸、イミノジ酢酸、３，３’−イミノジプロピオン酸、グルタミン酸等のモノアミン類からなる群から選択される。
【００１６】
【発明の実施の形態】
本発明は、前記の通り、光学活性アミノポリカルボン酸の製造における副反応を抑制することを目的として、該処理によりＥＤＤＳアーゼ活性を保持しながら、フマラーゼ活性を選択的に除去した微生物触媒を得ることを目的とする。したがって、フマル酸を原料とし、かつ該触媒を光学活性アミノポリカルボン酸製造に適用する限り、本発明の効果を得ることができる。
これら適用場面としては、具体的に、下記一般式〔２〕で示されるアミノ基を有する化合物とフマル酸の混合物から下記一般式〔１〕で示される光学活性アミノポリカルボン酸を製造する場合を挙げることができる：
【００１７】
【化３】

【００１８】
【化４】

【００１９】
式中、Ｒ_１およびＲ_２は、互いに同一でも異なっていてもよく、それぞれ水素原子（但し、Ｒ_１とＲ_２は共に水素原子であることはない）、置換または無置換のアルキル基、置換または無置換のシクロアルキル基、置換または無置換のアリール基、Ｒ_３およびＲ_４はＲ_１とＲ_２と同一またはＲ_１とＲ_２のアミノ基の少なくとも１個がその窒素原子を介してコハク酸のエチレン基の炭素原子と結合した構造を有する基等を表し、置換基はアミノ基、シアノ基、ニトロ基、ハロゲン、ヒドロキシル基、カルボキシル基、エーテル基等である。
【００２０】
一般式〔２〕で示されるアミノ基を有する化合物としては、例えば、エチレンジアミン、１，３−プロパンジアミン、２−メチル−１，３−プロパンジアミン、２−ヒドロキシ−１，３−プロパンジアミン、１，４−ブタンジアミン、１，５−ペンタンジアミン、１，６−ヘキサンジアミン、１，２−シクロヘキサンジアミン、１，３−シクロヘキサンジアミン、１，４−シクロヘキサンジアミン等の炭素数２〜６のアルカン−またはシクロアルカン−ジアミン類、１，２−フェニレンジアミン、１，３−フェニレンジアミン、１，４−フェニレンジアミン等のフェニレンジアミン類、ならびにグリシン、３−アミノプロピオン酸、２−アミノプロピオン酸、イミノジ酢酸、３，３’−イミノジプロピオン酸、グルタミン酸等のモノアミン類などを挙げることができ、エチレンジアミンが代表的である。
【００２１】
また、本発明で得られる一般式〔１〕で示される光学活性アミノポリカルボン酸の代表例としては、いずれも（Ｓ，Ｓ）−体のエチレンジアミン−Ｎ，Ｎ’−ジコハク酸、１，３−プロパンジアミン−Ｎ、Ｎ’−ジコハク酸、２−メチル−１，３−プロパンジアミン−Ｎ，Ｎ’−ジコハク酸、２−ヒドロキシ−１，３−プロパンジアミン−Ｎ，Ｎ’−ジコハク酸、１，４−ブタンジアミン−Ｎ，Ｎ’−ジコハク酸、１，５−ペンタンジアミン−Ｎ，Ｎ’−ジコハク酸、１，６−ヘキサンジアミン−Ｎ，Ｎ’−ジコハク酸、１，２−シクロヘキサンジアミン−Ｎ，Ｎ’−ジコハク酸、１，３−シクロヘキサンジアミン−Ｎ，Ｎ’−ジコハク酸、１，４−シクロヘキサンジアミン−Ｎ，Ｎ’−ジコハク酸、１，２−フェニレンジアミンジコハク酸、１，３−フェニレンジアミンジコハク酸および１，４−フェニレンジアミンジコハク酸等のアルカンジアミン−Ｎ，Ｎ’−ジコハク酸、シクロアルカンジアミン−Ｎ，Ｎ’−ジコハク酸またはフェニレンジアミン−Ｎ，Ｎ’−ジコハク酸；ならびに、いずれも（Ｓ）−体のアスパラギン酸−Ｎ−モノ酢酸、アスパラギン酸−Ｎ−モノプロピオン酸、アスパラギン酸−Ｎ−２−プロピオン酸、アスパラギン酸−Ｎ、Ｎ−ジ酢酸、アスパラギン酸−Ｎ、Ｎ−ジプロピオン酸、アスパラギン酸−Ｎ−２−グルタール酸などを挙げることができ、（Ｓ，Ｓ）−エチレンジアミン−Ｎ，Ｎ’−ジコハク酸が代表的である。
【００２２】
本発明のフマラーゼ活性除去処理の対象となる微生物は後記のとおりである。フマラーゼ活性除去処理は、培養により得られた菌体またはその処理物（菌体破砕物、菌体抽出液、抽出した粗・精製酵素、固定化した菌体または酵素、薬剤処理（安定化処理等）した菌体または酵素）に対して行うことができる。その他、培養終了後、培養液中の菌体に対して直接行うこともできる。また、フマラーゼ活性除去処理後に、別の処理を加えても差し支えない。
【００２３】
フマラーゼ活性の除去処理は、上記微生物菌体等を塩類の存在下にアルカリ水溶液に浸漬することにより行うことができる。また、該アルカリ水溶液としてはメタノール、エタノール等の水と自由に混和し得る有機溶媒や水と混和し得る範囲でその他の有機溶媒を併用したものでもよい。
塩類としては、本発明のアルカリ水溶液に溶解し得るものであれば、無機、有機、特に制限されない。例えば、ほう酸、りん酸、塩酸、硫酸、酢酸、蓚酸、前記フマル酸やマレイン酸、エチレンジアミン−Ｎ，Ｎ’−ジコハク酸等のナトリウム、カリウム、アンモニア、アミン類等の塩であり、アミン類としてはエチレンジアミン、プロパンジアミン、ブタンジアミン、ヘキサンジアミン等の炭素数２〜６のアルカンジアミン、トリエタノールアミン等のモノアミンが好適である。
【００２４】
塩類の濃度は５〜１０００ｍＭ、好ましくは１０〜５００ｍＭの範囲である。５ｍＭ未満では効果が充分でなく、１０００ｍＭを越えると効果が頭打ちとなる。
アルカリ水溶液のｐＨは８〜１０．５、好ましくは８．５〜１０、より好ましくは９〜９．５の範囲であり、上記の酸および塩基の割合を変えることにより所定のｐＨに調整することができる。中でも、ほう酸、りん酸もしくはＧｏｏｄの緩衝液等の使用は塩濃度およびｐＨの調整の両面から好適である。
【００２５】
処理温度は通常氷結温度〜５５℃の範囲である。処理時間は諸条件により異なるが、約１分〜約１ヶ月間であり、処理温度をより高温に設定することにより処理時間を短縮することができる。また、処理時間は延長されるものの、上記条件下、冷蔵保存中にフマラーゼ活性を除去することも可能である。
これらの処理操作は、回分、連続のいずれの方法でも行うことができる。
【００２６】
本発明の微生物としてはＥＤＤＳアーゼおよびフマラーゼ活性を有する微生物であればいずれも対象となる。例えば、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属、パラコッカス（Ｐａｒａｃｏｃｃｕｓ）属、スフィンゴモナス（Ｓｐｈｉｎｇｏｍｏｎａｓ）属およびブレブンジモナス（Ｂｒｅｖｕｎｄｉｍｏｎａｓ）属に属する細菌、さらに宿主としてエシェリヒア（Ｅｓｈｅｒｉｃｈｉａ）属またはロドコッカス（Ｒｈｏｄｏｃｏｃｃｕｓ）属に属する細菌を用い、これにＥＤＤＳアーゼをコードする遺伝子ＤＮＡを導入した形質転換体などを例として挙げることができる。
【００２７】
具体的には、Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．ＴＮ−１３１株（ＦＥＲＭ ＢＰ−５４１８）、Ｐａｒａｃｏｃｃｕｓ ｓｐ．ＴＮＯ−５株（ＦＥＲＭ ＢＰ−６５４７）、Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｐ．ＴＮ−２８株（ＦＥＲＭ ＢＰ−５４１９）、Ｂｒｅｖｕｎｄｉｍｏｎａｓ ｓｐ．ＴＮ−３０株（ＦＥＲＭＢＰ−５４１７）および同ＴＮ−３株（ＦＥＲＭ ＢＰ−５８８６）、さらに、宿主として大腸菌ＪＭ１０９株（Ｅｓｈｅｒｉｃｈｉａ ｃｏｌｉ ＡＴＣＣ５３３２３）、Ｒｈｏｄｏｃｏｃｃｕｓ ｒｈｏｄｏｃｈｒｏｕｓ ＡＴＣＣ１７８９５株およびＲｈｏｄｏｃｏｃｃｕｓ ｒｈｏｄｏｃｈｒｏｕｓ Ｊ−１株（ＦＥＲＭ ＢＰ−１４７８）を用いた形質転換体を挙げることができる。
【００２８】
上記微生物のうち、ＴＮ−１３１株、ＴＮＯ−５株、ＴＮ−２８株、ＴＮ−３０株、ＴＮ−３株は、本発明者らにより自然界から新たに分離され、上記番号にて通産省工業技術院生命工学工業技術研究所に寄託されている。これらの菌株の菌学的性質は以下に示す通りである。
【００２９】

【００３０】

【００３１】

【００３２】

【００３３】
上記菌学的性質を、Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ Ｖｏｌ．１（１９８４）および Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｄｅｔｅｒｍｉｎａｔｉｖｅ Ｂａｃｔｅｒｉｏｌｏｇｙ ９版（１９９４）により分類するとＴＮ−１３１株はシュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属に、Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆＳｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ Ｖｏｌ．１（１９８４）により分類するとＴＮＯ−５株はパラコッカス（Ｐａｒａｃｏｃｃｕｓ）属に、Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｄｅｔｅｒｍｉｎａｔｉｖｅ Ｂａｃｔｅｒｉｏｌｏｇｙ ９版（１９９４）および Ｍｉｃｒｏｂｉｏｌ． Ｉｍｍｕｎｏｌ．３４，９９（１９９０）により分類するとＴＮ−２８株はスフィンゴモナス（Ｓｐｈｉｎｇｏｍｏｎａｓ）属に、また、Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｄｅｔｅｒｍｉｎａｔｉｖｅ Ｂａｃｔｅｒｉｏｌｏｇｙ ９版（１９９４）ならびにＩｎｔ． Ｊ． Ｓｙｓｔ． Ｂａｃｔｅｒｉｏｌ． ４４，４９９（１９９４）により分類するとＴＮ−３０株およびＴＮ−３株はブレブンジモナス（Ｂｒｅｖｕｎｄｉｍｏｎａｓ）属に属する細菌と同定される。尚、ＴＮ−３株はディミヌタ（ｄｉｍｉｎｕｔａ）種であることが確認されている。
【００３４】
大腸菌 ＪＭ１０９株（Ｅｓｈｅｒｉｃｈｉａ ｃｏｌｉ ＡＴＣＣ５３３２３）、Ｒｈｏｄｏｃｏｃｃｕｓ ｒｈｏｄｏｃｈｒｏｕｓ ＡＴＣＣ１７８９５株は公知であり、アメリカンタイプカルチャーコレクション（ＡＴＣＣ）から容易に入手することができる。これらの菌株を宿主として、ＴＮ−３株のＥＤＤＳアーゼ活性を有するタンパク質をコードする遺伝子ＤＮＡを含むプラスミドｐＥＤＳ０２０およびｐＳＥ００１を導入した形質転換体が、Ｅ． ｃｏｌｉ ＪＭ１０９／ｐＥＤＳ０２０（ＦＥＲＭ ＢＰ−６１６１；１９９７年１１月１０日に国際寄託に移管）およびＲｈｏｄｏｃｏｃｃｕｓ ｒｈｏｄｏｃｈｒｏｕｓＡＴＣＣ１７８９５／ｐＳＥ００１（ＦＥＲＭ ＢＰ−６５４８；１９９８年１０月１５日に国際寄託に移管）として、それぞれ通産省工業技術院生命工学工業技術研究所に寄託されている。以下に、これら形質転換体の作製方法を示す。
【００３５】
（１）ＴＮ−３株染色体ＤＮＡの調製
ＴＮ−３株を１００ｍｌのＥＤＤＳ培地（０．２％エチレンジアミン−Ｎ，Ｎ’−ジコハク酸、０．２％グルコース、０．１％バクトイーストエキス、０．０５％ポリペプトン、０．２８％硫酸ナトリウム、０．１％硫酸マグネシウム・７Ｈ_２Ｏ、２．５％（ｖ／ｖ）の１Ｍりん酸緩衝液（ｐＨ７．０）、０．５％（ｖ／ｖ）金属塩混合物溶液（１００ｍｌ中の組成；塩化マグネシウム・６Ｈ_２Ｏ８ｇ，塩化カルシウム０．８ｇ，硫酸マンガン・４Ｈ_２Ｏ ０．６ｇ，塩化第二鉄・６Ｈ_２Ｏ０．１２ｇ，硫酸亜鉛０．０６ｇ））中、３０℃にて４日間振とう培養した後集菌し、この菌体をＳａｌｉｎｅ−ＥＤＴＡ溶液（０．１Ｍ ＥＤＴＡ、１５Ｍ ＮａＣｌ（ｐＨ８．０））４ｍｌに懸濁しリゾチーム８ｍｇを加えて３７℃で１時間振とうした後凍結する。次に１０ｍｌのＴｒｉｓ−ＳＤＳ液（１％ＳＤＳ、０．１ＭＮａＣｌ、０．１Ｍ Ｔｒｉｓ（ｐＨ９．０））を穏やかに振とうしながら加え、さらにプロテイナーゼＫ（メルク社製）（終濃度１ｍｇ）を加え３７℃で１時間振とうする。次に等量のＴＥ飽和フェノールを加え撹拌後（ＴＥ：１０ｍＭ Ｔｒｉｓ、１ｍＭ ＥＤＴＡ（ｐＨ８．０））遠心し、上層を取り２倍量のエタノールを加えた後、ガラス棒でＤＮＡを巻きとり、９０％、８０％、７０％のエタノールで順次フェノールを取り除く。次にＤＮＡを３ｍｌのＴＥ緩衝液に溶解させ、リボヌクレアーゼＡ溶液（１００℃、１５分間の熱処理済み）を１０ｍｇ／ｍｌになるように加え３７℃で３０分間振とうする。さらにプロテイナーゼＫを加え３７℃で３０分間振とうした後、等量のＴＥ飽和フェノールを加え遠心し上層と下層に分離する。上層についてこの操作を２回繰り返した後、同量のクロロフォルム（４％イソアミルアルコール含有）を加え同様の抽出操作を繰り返す（以後この操作をフェノール処理と呼ぶ）。その後上層に２倍量のエタノールを加えガラス棒でＤＮＡを巻き取り回収し、染色体ＤＮＡ標品を得る。
【００３６】
（２）精製酵素の調製
ＴＮ−３株を２リットルのＥＤＤＳ培地中、３０℃にて４日間振とう培養した後集菌し、１０ｍＭリン酸ナトリウム緩衝液（ｐＨ８．０、１ｍＭジチオスレイトール含有）１００ｍｌに懸濁し、超音波破砕機で菌体を破砕し、１２，０００ｒｐｍで２０分間遠心分離する。得られた上澄に３０％飽和となるように硫酸アンモニウムを添加し、４℃にて３０分間静置後遠心分離する。得られた上澄に６０％飽和となるように硫酸アンモニウムを添加し４℃にて３０分間静置後遠心分離する。得られた沈殿を１０ｍｌの１０ｍＭリン酸ナトリウム緩衝液（ｐＨ８．０，１ｍＭジチオスレイトール含有）に溶解し、部分精製酵素液とする。
【００３７】
この部分精製酵素液をイオン交換クロマトグラフィーでさらに精製する。すなわち、１ｍＭジチオスレイトールを含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ８．０）で緩衝化したＤＥＡＥ−Ｓｅｐｈａｃｒｙｌ（ファルマシア社製）を充填したカラム（φ１０ｍｍ × ２０ｃｍ）に、部分精製酵素液を流し吸着させた。４０ｍｌの同緩衝液でカラムを洗浄した後、０〜０．６Ｍ ＫＣｌの直線勾配で本酵素を溶出させ、２ｍｌづつ分画する。ＥＤＤＳアーゼ活性の認められた画分を集めて精製酵素液とする。この画分をＳＤＳ−ポリアクリルアミドゲル電気泳動により分析したところ分子量約６万のほぼ均一な一本のバンドが検出される。
【００３８】
（３）精製酵素のＮ末端アミノ酸配列および内部ペプチドのアミノ酸配列の分析（２）の工程で得られた精製酵素を直接、あるいは ｔｒｙｐｓｉｎで消化後ＳＤＳ−ポリアクリルアミドゲル電気泳動でポリペプチドを分画し、ＰＶＤＦメンブレン（ＩｍｍｏｂｉｌｏｎＰｓｑ（ミリポア社製））にエレクトロブロッティングする。ブロッティングしたメンブレンをクマシーブリリアントブルーで染色し、染色されたバンドを切り取りアミノ酸配列の分析に供する。アミノ酸配列の分析は島津製作所社製 ＰＳＱ−１型アミノ酸分析装置により行う。結果を以下に示す。
【００３９】
ａ）無処理酵素のＮ末端アミノ酸配列：（分子量約６万）；
Ｘａａ−Ｔｈｒ−Ｐｒｏ−Ｈｉｓ−Ａｓｎ−Ｐｒｏ−Ａｓｐ−Ａｌａ
（配列番号６；ここでＸａａはＭｅｔまたは欠失を示す。）
ｂ）ｔｒｙｐｓｉｎ 部分分解物：（分子量約５万）；
Ｇｌｕ−Ｉｌｅ−Ｇｌｙ−Ｓｅｒ−Ｖａｌ−Ｇｌｙ−Ｌｙｓ−Ｍｅｔ−Ｇｌｕ−Ｉｌｅ−Ｇｌｙ−Ａｒｇ−Ｘａａ−Ａｌａ−Ａｓｎ−Ａｓｐ−Ｌｅｕ−Ａｒｇ−Ａｓｎ−Ａｒｇ
（配列番号７；ここでＸａａは未同定アミノ酸残基を示す。）
【００４０】
ｃ）ｔｒｙｐｓｉｎ 部分分解物：（分子量約１万）；
Ａｌａ−Ｓｅｒ−Ｇｌｙ−Ａｌａ−Ｌｙｓ−Ａｌａ−Ｐｒｏ−Ｇｌｕ−Ｐｈｅ−Ｇｌｎ−Ｇｌｕ−Ｌｅｕ−Ｔｙｒ−Ａｓｐ−Ｐｈｅ−Ｇｌｕ−Ａｌａ−Ａｌａ−Ｘａａ−Ｌｅｕ−Ｘａａ−Ｌｅｕ
（配列番号８；ここでＸａａは未同定アミノ酸残基を示す。）
〔括弧内の分子量は分画したペプチドの分子量を示す。〕
【００４１】
（４）プローブの調製
（３）の工程で得られたアミノ酸配列情報を基に配列番号４および５に示されるような合成ＤＮＡを作製してプライマー（プライマー＃１、＃２）とし、（１）の工程で得られたＴＮ−３株の染色体ＤＮＡを鋳型としてポリメラーゼ連鎖反応を行う。
すなわち、ＴＮ−３株染色体ＤＮＡ１μｌ、１０倍濃度の反応緩衝液１０μｌ、１０ｍＭｄＮＴＰ４μｌ、プライマー＃１、＃２各々１μｌ（１００ｐｍｏｌ濃度相当）、ＥｘＴａｑ （宝酒造社製）１μｌを加えて１００μｌとした。この溶液について、９５℃、３０秒間（変性ステップ）、５５℃、３０秒間（アニーリングステップ）、７２℃、２分間（伸長ステップ）のインキュベーションを３０サイクル行う。反応終了後、クロロフォルム抽出を３回行い、エタノール沈殿により増幅されたＤＮＡを回収する。これを１．０％アガロース電気泳動で分離後、ＴＮ−３株のＥＤＤＳアーゼ遺伝子をコードすると考えられる約３００ｂｐのＤＮＡ断片を得る。こうして得られたＤＮＡ断片を ＤＩＧ ＤＮＡ Ｌａｂｅｌｉｎｇ ｋｉｔ （ベーリンガー・マンハイム社製）を用いて標識し、プローブとする。
【００４２】
（５）ＤＮＡライブラリーの作製
ＴＮ−３株染色体ＤＮＡ１０μｌに１０倍濃度制限酵素反応用緩衝液５μｌ、滅菌水３３μｌ、制限酵素 ＫｐｎＩ ２μｌを加え、３７℃にて１６時間反応させた後エタノール沈殿によりＤＮＡを回収する。アガロース電気泳動を行い、６．５Ｋｂから５．５ＫｂのＤＮＡ断片をゲルから切り出し ＤＮＡ ＰＲＥＰ（ダイヤトロン社製）を用いて回収する。このＤＮＡ断片をライゲーションキット（宝酒造社製）を用いて大腸菌ベクターｐＵＣ１８のＫｐｎＩ部位に挿入し、組換え体ＤＮＡライブラリーを作製する。
【００４３】
ライゲーションに用いたｐＵＣ１８断片は次のように作製する。ｐＵＣ１８保存液２μｌに対し、１０倍濃度制限酵素用緩衝液５μｌ、滅菌水４０μｌ、制限酵素 ＫｐｎＩ ３μｌを加え、３７℃で２時間反応後、フェノール処理、エタノール沈殿させた後乾燥して５０μｌの滅菌水に溶解させる。さらにアルカリフォスファターゼ（宝酒造株式会社）１μｌ、１０倍濃度緩衝液１０μｌ、滅菌水３９μｌを加え６５℃で反応後フェノール処理、エタノール沈殿を行い乾燥して滅菌水に溶解させる。
【００４４】
（６）大腸菌形質転換体の作製および組換え体ＤＮＡの選別
大腸菌ＪＭ１０９株をＬＢ培地（１％バクトトリプトン、０．５％バクトイーストエキス、０．５％ＮａＣｌ）１ｍｌに接種し３７℃、５時間好気的に前培養し、この培養物１００ｍｌをＳＯＢ培地（２％バクトトリプトン、０．５％バクトイーストエキス、１０ｍＭ ＮａＣｌ、２．５ｍＭ ＫＣｌ、１ｍＭ ＭｇＳＯ_４、１ｍＭ ＭｇＣｌ_２）４０ｍｌに加え、１８℃で２０時間培養する。この培養物を遠心分離により集菌した後、冷ＴＦ溶液（２０ｍＭ ＰＩＰＥＳ−ＫＯＨ（ｐＨ６．０）、２００ｍＭ ＫＣｌ、１０ｍＭ ＣａＣｌ_２、４０ｍＭ ＭｎＣｌ_２）を１３ｍｌ加え、０℃で１０分間放置後、再度遠心する。上澄を除いた後、沈殿した大腸菌を冷ＴＦ溶液３．２ｍｌに懸濁し、０．２２ｍｌのジメチルスルフォキシドを加え０℃で１０分間放置する。こうして作製したコンピテントセル２００μｌに工程（５）で作製した組換え体プラスミドを含有する溶液（ＤＮＡライブラリー）を１０μｌ加え、０℃で３０分放置後、４２℃で３０秒間ヒートショックを与え０℃で２分間冷却後、ＳＯＣ培地（２０ｍＭグルコース、２％バクトトリプトン、０．５％バクトイーストエキス、１０ｍＭ ＮａＣｌ、２．５ｍＭ ＫＣｌ、１ｍＭ ＭｇＳＯ_４、１ｍＭ ＭｇＣｌ_２）１ｍｌを添加して３７℃にて１時間振とう培養する。これを２００μｌずつアンピシリン１００μｇ／ｍｌ含有のＬＢ寒天培地にまき、３７℃で培養する。寒天培地上に生育した形質転換体コロニーについてコロニーハイブリダイゼーション法にてＥＤＤＳアーゼ遺伝子を持つ形質転換体を選別する。すなわち、寒天培地上に生育した形質転換体をナイロンメンブレン（バイオダインＡ：日本ポール社製）上に移し、菌体を溶かしてＤＮＡを固定した後、これを工程（４）で作製したプローブ（約３００ｂｐ）で処理し、ＤＩＧ Ｌｕｍｉｎｅｓｃｅｎｔ Ｄｅｔｅｃｔｉｏｎ Ｋｉｔ （ベーリンガー・マンハイム社製）を用い、目的の組換え体ＤＮＡを持つコロニーを選択する。
【００４５】
（７）組換えプラスミドの調製
工程（６）で選択した形質転換体を１００ｍｌのＬＢ培地にて３７℃で一晩培養し、集菌後滅菌水により洗浄し、溶液Ｉ（２ｍＭグルコース、１０ｍＭ ＥＤＴＡ、２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０））を５ｍｌ、リゾチームを２５ｍｇ加え、０℃で３０分間放置する。溶液ＩＩ（１Ｎ ＮａＯＨ、５％ＳＤＳ）を１０ｍｌ加え０℃で５分間放置し、溶液ＩＩＩ（３Ｍ酢酸ナトリウム（ｐＨ４．８））を７．５ｍｌ加え０℃で３０分間放置する。これを遠心し、その上澄に５０ｍｌのエタノールを加え、さらに遠心し上澄を取り除き５ｍｌの溶液ＩＶ（１０ｍＭ酢酸ナトリウム、５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０））とリボヌクレアーゼＡ溶液（１０ｍｇ／ｍｌ）２．５μｌ加え室温で２０分間放置する。これに１２ｍｌのエタノールを加え、遠心によりプラスミドを回収、７０％エタノールでリンスし、乾燥後０．４ｍｌの滅菌水に溶解する。こうして得られた組換え体プラスミドをｐＥＤＳ００１と名付ける。
【００４６】
（８）制限酵素地図の作製およびＥＤＤＳアーゼ遺伝子領域の特定
工程（７）で得られたプラスミドｐＥＤＳ００１を数種の制限酵素を用いて切断し制限酵素地図を作製する（図１）。さらに、通常行われるようにサブクローニングを行う。すなわち、ｐＥＤＳ００１を制限酵素ＫｐｎＩとＢａｍＨＩとで切断し、ｐＵＣ１８を同制限酵素で切断したものとライゲーションし、大腸菌ＪＭ１０９株を形質転換することにより、約３．９Ｋｂの断片が挿入されたプラスミド（ｐＥＤＳ００２）（図２）と約２．６Ｋｂの断片が挿入されたプラスミド（ｐＥＤＳ００３）（図３）を得る。各々のプラスミドについて制限酵素ＢａｍＨＩ、ＥｃｏＲＩ、ＳａｃＩ、ＳａｌＩなどで切断後、アガロース電気泳動を行い、サザンハイブリダイゼーション法によりプローブがハイブリダイズする断片を特定する。
【００４７】
（９）塩基配列の決定
工程（８）で特定された領域周辺の塩基配列をファルマシア社蛍光シーケンサＡＬＦＩＩを用いて決定する。その結果、配列番号１に示される塩基配列が得られ、配列番号２に示されるアミノ酸配列を持つオープンリーディングフレームが見いだされる。アミノ酸配列データーベース ＮＢＲＦ（Ｎａｔｉｏｎａｌ Ｂｉｏｍｅｄｉｃａｌ Ｒｅｓｅａｒｃｈ Ｆｏｕｎｄａｔｉｏｎ）との比較の結果本遺伝子は、ｄｅｌｔａ−ｃｒｙｓｔａｌｌｉｎあるいは ａｒｇｉｎｉｎｏｓｕｃｃｉｎａｔｅ ｌｙａｓｅと２０〜３０％のホモロジーを有しており、両酵素にはともにフマル酸とアミン（アミノ酸）の縮合あるいは分解反応を触媒する活性が知られている。また、このオープンリーディングフレームの塩基配列を配列番号３に示す。
【００４８】
（１０）プラスミド ｐＥＤＳ０２０と大腸菌形質転換体の作製、および形質転換体のＥＤＤＳアーゼ活性
工程（８）で得られたＥＤＤＳアーゼ遺伝子を有する組換え体プラスミドｐＥＤＳ００３２μｌに対し、１０倍濃度制限酵素用緩衝液２μｌ、滅菌水１５μｌ、制限酵素ＫｐｎＩ１μｌを添加し、３７℃にて２時間反応させる。エタノール沈殿によりプラスミドを回収し、乾燥後１７μｌの滅菌水、２μｌの１０倍濃度制限酵素緩衝液、制限酵素ＢａｍＨＩ１μｌを添加して３７℃にて２時間反応させる。この反応液からアガロース電気泳動により約２．６Ｋｂの断片を調製し、大腸菌ベクターｐＵＣ１１９に挿入する。作製したライゲーション溶液を用いて大腸菌ＪＭ１０９を形質転換して目的のプラスミドを得る。ここで作製したプラスミドをｐＥＤＳ０２０（図４）と、また形質転換体をＪＭ１０９／ｐＥＤＳ０２０と名付ける。
【００４９】
ＪＭ１０９／ｐＥＤＳ０２０をアンピシリン５０ｍｇ／ｌ含有するＬＢ培地１ｍｌに接種して３７℃にて８時間振とう培養後、全量を４０ｍｌのＬＢ培地（５０ｍｇ／ｌアンピシリン、１ｍＭ ｉｓｏｐｒｏｐｙｌ−β−ｔｈｉｏｇａｌａｃｔｏｓｉｄｅを含有）に接種し３７℃、３０時間培養した。得られた培養物を１０ｍＭリン酸ナトリウム緩衝液（ｐＨ８．０）で洗浄後、２ｍｌの同緩衝液に懸濁する。得られた菌体懸濁液の一部を３４２ｍＭフマル酸と１７１ｍＭエチレンジアミンを含むｐＨ８．０の水溶液５０ｍｌに懸濁し２４時間反応させる。反応液から菌体を遠心分離により除いた後、ＨＰＬＣを用いて生成したエチレンジアミン−Ｎ，Ｎ’−ジコハク酸を分析する（ＷＡＫＯＳＩＬ ５Ｃ８（和光純薬社製）〔溶出液；１０ｍＭ水酸化テトラ−ｎ−ブチルアンモニウムと０．４ｍＭＣｕＳＯ_４を含む５０ｍＭリン酸、ｐＨ２〕）。その結果、５０ｍＭのＳ，Ｓ−エチレンジアミン−Ｎ，Ｎ’−ジコハク酸が生成していた。
【００５０】
（１１）プラスミドｐＳＥ００１の作製
工程（１０）で作製したプラスミドｐＥＤＳ０２０、２μｌに対し、１０倍濃度制限酵素用緩衝液２μｌ、滅菌水１５μｌ、制限酵素ＸｈｏＩ １μｌを添加し、３７℃にて２時間反応させる。エタノール沈殿によりプラスミドを回収し、乾燥後１５μｌの滅菌水、２μｌの１０倍濃度ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔ用緩衝液、２μｌの１０ｍＭ ｄＮＴＰ溶液、１μｌの ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを添加して３７℃にて２時間反応させる。エタノール沈殿によりＤＮＡ断片を回収し、乾燥後８μｌの滅菌水、１μｌのＸｂａＩリンカー溶液、１６μｌの Ｌｉｇａｔｉｏｎ ｋｉｔ （宝酒造社製）溶液Ａおよび４μｌの溶液Ｂを添加して１６℃にて４時間反応させた後 ＪＭ１０９に形質転換させる。得られた形質転換体よりプラスミドを調製して、ｐＥＤＳ０２０のＸｈｏＩ部位がＸｂａＩ部位に変換されたプラスミドを得る。得られたプラスミド２μｌに対し、１０倍濃度制限酵素用緩衝液２μｌ、滅菌水１５μｌ、制限酵素ＥｃｏＲＶ１μｌを添加し、３７℃にて２時間反応させる。エタノール沈殿によりＤＮＡ断片を回収し、乾燥後８μｌの滅菌水、１μｌの Ｓｓｅ８３８７Ｉリンカー溶液、１６μｌのＬｉｇａｔｉｏｎ ｋｉｔ （宝酒造社製）溶液Ａおよび４μｌの溶液Ｂを添加して１６℃にて４時間反応させた後ＪＭ１０９に形質転換させる。得られた形質転換体よりプラスミドを調製し、ＥｃｏＲＶ部位がＳｓｅ８３８７Ｉ部位に変換されたプラスミド（ｐＥＤＳ０２７）を得る。得られたプラスミド２μｌに対し、１０倍濃度制限酵素用緩衝液２μｌ、滅菌水１４μｌ、制限酵素ＸｂａＩ１μｌおよびＳｓｅ８３８７Ｉ １μｌを添加し、３７℃にて２時間反応させる。その後、０．７％アガロース電気泳動で分離し１．７Ｋｂのバンドを回収しロドコッカス細菌用の強力なプロモーター活性を有するプラスミドｐＳＪ０３４のＸｂａＩ−Ｓｓｅ８３８７Ｉ部位に挿入し、組換え体プラスミドｐＳＥ００１を作製する（図５）。ｐＳＪ０３４はプラスミドｐＳＪ０２３（特願平９−６５６１８号明細書参照）より図６に示した工程により作製する。ｐＳＪ０２３は形質転換体 Ｒｈｏｄｏｃｏｃｃｕｓ ｒｈｏｄｏｃｈｒｏｕｓ ＡＴＣＣ１２６７４／ｐＳＪ０２３（ＦＥＲＭ ＢＰ−６２３２；１９９８年１月２１日に国際寄託に移管）として工業技術院生命工学工業技術研究所に寄託されている。
【００５１】
（１２）Ｒｈｏｄｏｃｏｃｃｕｓ属細菌の形質転換および形質転換体のＥＤＤＳアーゼ活性
Ｒｈｏｄｏｃｏｃｃｕｓ ｒｈｏｄｏｃｈｒｏｕｓ ＡＴＣＣ１７８９５株の対数増殖期の細胞を遠心分離器により集菌し、氷冷した滅菌水にて３回洗浄し、滅菌水に懸濁する。プラスミドｐＳＥ００１ １μｌと菌体懸濁液１０μｌを混合し、氷冷する。キュベットにＤＮＡと菌体の懸濁液を入れ、遺伝子導入装置Ｇｅｎｅ Ｐｕｌｓｅｒ（ＢＩＯ ＲＡＤ）により２．０ＫＶ、２００ＯＨＭＳで電気パルス処理を行う。電気パルス処理液を氷冷下、１０分間静置し、３７℃で１０分間ヒートショックを行い、ＭＹＫ培地（０．５％ポリペプトン、０．３％バクトイーストエキス、０．３％バクトモルトエキス、０．２％Ｋ_２ＨＰＯ_４、０．２％ＫＨ_２ＰＯ_４）５００ｍｌを加え、３０℃、５時間静置した後、５０ｍｇ／ｌカナマイシン含有ＭＹＫ寒天培地に塗布し、３０℃、３日間培養する。得られた形質転換体をＡＴＣＣ１７８９５／ｐＳＥ００１と名づける。
【００５２】
こうして作製した形質転換体をＭＹＫ培地（５０ｍｇ／ｌカナマイシン含有）１０ｍｌに接種し、３０℃で７２時間振とう培養する。このうち１ｍｌを培地１００ｍｌ（１．５％グルコース、１％グルタミン酸ナトリウム、０．１％バクトイーストエキス、０．０５％ＫＨ_２ＰＯ_４、０．０５％Ｋ_２ＨＰＯ_４、０．０５％硫酸マグネシウム、ｐＨ７．２、５０ｍｇ／ｌカナマイシン含有）に接種し、３０℃で６０時間培養する。次に、得られた菌体を用い、工程（１０）記載の方法にて反応、分析を行ったところ、３６ｍＭのＳ，Ｓ−エチレンジアミン−Ｎ，Ｎ’−ジコハク酸が生成していた。
【００５３】
式［１］で示される光学活性アミノポリカルボン酸を製造するための反応は、水または緩衝剤溶液（例えばりん酸緩衝液、炭酸緩衝液またはほう酸緩衝液）中で、上述した本発明の微生物株またはその処理物（即ち、菌体破砕物、菌体抽出液、菌体から抽出された粗酵素または精製酵素、固定化した菌体または酵素、安定化剤等の薬剤で処理された菌体または酵素から選択される）を、フマル酸と式［２］で示されるアミノ基含有化合物との混合物と接触させることによって行われる。
【００５４】
一般に、この反応は、０〜５０℃、好ましくは５〜３５℃の温度、および５〜１１、好ましくは６〜１０のｐＨで行うことができる。フマル酸および式［２］で示されるアミノ基含有化合物の濃度は使用する反応温度やｐＨに応じて変化し得るが、それらの濃度は各々０．１％〜飽和濃度の範囲であり得る。また、使用する微生物またはその処理物の量は、通常、基質の量に基づいて０．０１〜５．０％である。反応は回分式または連続式のいずれかで行うことができる。
【００５５】
反応の完結時に反応混合物から生成アミノポリカルボン酸を単離するために、微生物の除去、濃縮、クロマトグラフィー（例えばＨＰＬＣ）、結晶化などの公知の技術を使用することができる。
アミノポリカルボン酸の製造の具体的手順に関しては、特開平９−１４０３９０号公報または対応の米国特許第５７０７８３６号明細書に記載される手順と同様の仕方で行うことができる。これらの特許公開公報、米国特許の内容は本明細書に参考として含めるものとする。
【００５６】
【実施例】
次に、本発明を以下の実施例により具体的に説明する。
＜実施例１＞
（１）培養
Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ． ＴＮ−１３１株、Ｐａｒａｃｏｃｃｕｓｓｐ．ＴＮＯ−５株、Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｐ．ＴＮ−２８株、Ｂｒｅｖｕｎｄｉｍｏｎａｓ ｓｐ． ＴＮ−３０株および同ＴＮ−３株を斜面培地から１白金耳取り、下記の培地に接種し、３０℃、４日間好気的に振とう培養した。
【００５７】
培地組成（ｐＨ７．５，１００ｍｌ）：
エチレンジアミン−Ｎ，Ｎ’−ジコハク酸 ０．２ｇ
グルコース ０．２ｇ
酵母エキス ０．１ｇ
ポリペプトン ０．０５ｇ
硫酸マグネシウム・７Ｈ_２Ｏ ０．１ｇ
硫酸ナトリウム ０．２８ｇ
燐酸緩衝液 ２５ｍＭ
金属塩混合物溶液＊ ０．５ｍｌ
［＊金属塩混合物溶液（１００ｍｌ）： 塩化マグネシウム・６Ｈ_２Ｏ ８ｇ，塩化カルシウム ０．８ｇ，硫酸マンガン・４Ｈ_２Ｏ ０．６ｇ，塩化第二鉄・６Ｈ_２Ｏ ０．１２ｇ，硫酸亜鉛 ０．０６ｇ］
【００５８】
（２）フマラーゼ活性除去処理
菌体培養液を遠心管に取り１０，０００ｒｐｍ、５℃、１５分間の遠心分離にて集菌した後、５０ｍＭほう酸緩衝液（ｐＨ９．２）で２回洗浄し、同緩衝液で５ｍｇ乾燥菌体／ｍｌに希釈し、４５℃、６時間、水浴中で処理を行った。なお、２時間経過後にサンプリングを行った。
【００５９】
（３）ＥＤＤＳアーゼおよびフマラーゼの活性測定
上記フマラーゼ除去処理を施した菌体は、ＥＤＤＳアーゼおよびフマラーゼの活性測定を行い、未処理菌体の活性を１００％として残存活性を求めた。
ＥＤＤＳアーゼの活性測定は６８４ｍＭフマル酸、３４２ｍＭエチレンジアミン、３４２ｍＭ水酸化マグネシウムを含み、６Ｎ ＮａＯＨでｐＨ８．５に調整した反応液を用い、処理液の１／１０の菌体濃度にて３０℃、一昼夜反応させ、生成する（Ｓ，Ｓ）−エチレンジアミン−Ｎ，Ｎ’−ジコハク酸（Ｓ，Ｓ−ＥＤＤＳという）を下記操作にて定量した。
【００６０】
フマラーゼの活性測定は６８．４ｍＭ Ｌ−リンゴ酸ナトリウム、５０ｍＭほう酸緩衝液を含み、６Ｎ ＮａＯＨでｐＨ８．５に調整した反応液を用い、処理液の１／１０の菌体濃度にて３０℃、一昼夜反応させ、生成するフマル酸を下記操作にて定量した。
【００６１】
反応生成物の定量：
Ｓ，Ｓ−ＥＤＤＳ、フマル酸およびＬ−リンゴ酸の分析は、反応液中の不溶物を１５，０００ｒｐｍ、５℃、５分間の遠心分離にて除去した後、液体クロマトグラフィーにて行った。定量用カラムとしては ＷＡＫＯＳＩＬ ５Ｃ８（和光純薬）〔溶出液；１０ｍＭ 水酸化テトラ−ｎ−ブチルアンモニウムと０．４ｍＭ ＣｕＳＯ_４を含む５０ｍＭリン酸、ｐＨ２〕を、また光学分割カラムとしてＭＣＩ ＧＥＬ ＣＲＳ １０Ｗ（三菱化学社製）〔溶出液：１０ｍＭ ＣｕＳＯ_４〕を使用した。
（４）結果
【００６２】
【表１】

【００６３】
＜実施例２＞
（１）形質転換体の培養
Ｅｓｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＥＤＳ０２０
斜面培地から１白金耳とり、５０ｍｇ／ｌアンピシリンを含有するＬＢ培地（１％バクトトリプトン、０．５％バクトイーストエキス、０．５％ＮａＣｌ）に接種して３７℃にて８時間振とう培養した。これを、ＬＢ培地（５０ｍｇ／ｌアンピシリン、１ｍＭ ｉｓｏｐｒｏｐｙｌ−ｂ−ｇａｌａｃｔｏｓｉｄｅを含有）に、２．５％量接種し、３７℃、３０時間、好気的に振とう培養した。
【００６４】
Ｒｈｏｄｏｃｏｃｃｕｓ ｒｈｏｄｏｃｈｒｏｕｓ ＡＴＣＣ１７８９５／ｐＳＥ００１
斜面培地から１白金耳とり、５０ｍｇ／ｌカナマイシンを含有するＭＹＫ培地（０．５％ポリペプトン、０．３％バクトイーストエキス、０．３％バクトモルトエキス、０．２％Ｋ_２ＨＰＯ_４、０．２％ＫＨ_２ＰＯ_４）に接種し、３０℃、７２時間、振とう培養した。これを、ＧＧＹ培地（１．５％グルコース、１％グルタミン酸ナトリウム、０．１％バクトイーストエキス、０．０５％Ｋ_２ＨＰＯ_４、０．０５％ＫＨ_２ＰＯ_４、０．０５％硫酸マグネシウム、５０ｍｇ／ｌカナマイシン、ｐＨ７．２）に１％量接種し、３０℃で６０時間、好気的に振とう培養した。
【００６５】
（２）フマラーゼ活性除去処理
実施例１と同様。
（３）ＥＤＤＳアーゼおよびフマラーゼの活性測定
実施例１と同様。
（４）結果
【００６６】
【表２】

【００６７】
＜実施例３＞
（１）形質転換体の培養
Ｅｓｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＥＤＳ０２０を実施例２と同様に培養した。
（２）グルタルアルデヒド処理およびフマラーゼ活性除去処理
菌体培養液を遠心管に取り１０，０００ｒｐｍ、５℃、１５分間の遠心分離にて集菌した後、５０ｍＭほう酸緩衝液（ｐＨ９．２）で２回洗浄した。この菌体を、最終濃度１ｍＭのグルタルアルデヒドを含む同緩衝液中で、最終濃度５ｍｇ乾燥菌体／ｍｌにて、４℃、２時間処理した後、１００ｍＭほう酸緩衝液（ｐＨ９．２）で２回洗浄し、同緩衝液で５ｍｇ乾燥菌体／ｍｌに希釈した。このグルタルアルデヒド処理菌体と、同濃度に希釈した未処理菌体に対して、４５℃、６時間、水浴中でフマラーゼ活性除去処理を行った。
（３）ＥＤＤＳアーゼおよびフマラーゼの活性測定
実施例１と同様。
（４）結果
【００６８】
【表３】

【００６９】
＜実施例４＞
（１）形質転換体の培養
実施例３と同様。
（２）フマラーゼ活性除去処理
菌体培養液を遠心管に取り１０，０００ｒｐｍ、５℃、１５分間の遠心分離にて集菌した後、６Ｎ ＮａＯＨまたは５Ｎ硫酸にて表４に記載のｐＨに調整した５０ｍＭほう酸緩衝液で２回洗浄し、各々の緩衝液で５ｍｇ乾燥菌体／ｍｌに希釈し、４５℃、２時間、水浴中で処理を行った。
（３）ＥＤＤＳアーゼおよびフマラーゼの活性測定
実施例１と同様。
（４）結果
【００７０】
【表４】

【００７１】
＜実施例５＞
（１）形質転換体の培養
実施例３と同様。
（２）フマラーゼ活性除去処理
菌体培養液を遠心管に取り１０，０００ｒｐｍ、５℃、１５分間の遠心分離にて集菌した後、表５に記載の塩溶液（各々、６Ｎ ＮａＯＨまたは５Ｎ硫酸にてｐＨを９．２に調整した）で２回洗浄し、各々の塩溶液で５ｍｇ乾燥菌体／ｍｌに希釈し、４５℃、２時間、水浴中で処理を行った。
（３）ＥＤＤＳアーゼおよびフマラーゼの活性測定
実施例１と同様。
（４）結果
【００７２】
【表５】

【００７３】
＜実施例６＞
（１）形質転換体の培養
実施例３と同様。
（２）フマラーゼ活性除去処理
菌体培養液を遠心管に取り１０，０００ｒｐｍ、５℃、１５分間の遠心分離にて集菌した後、１００ｍＭほう酸緩衝液（ｐＨ９．２）で２回洗浄し、同緩衝液で５ｍｇ乾燥菌体／ｍｌに希釈し、表６記載の温度、時間にて水浴中で処理を行った。
（３）ＥＤＤＳアーゼおよびフマラーゼの活性測定
実施例１と同様。
（４）結果
【００７４】
【表６】

【００７５】
＜実施例７＞
（１）形質転換体の培養
実施例３と同様。
、２）フマラーゼ活性除去処理
菌体培養液を遠心管に取り１０，０００ｒｐｍ、５℃、１５分間の遠心分離にて集菌した後、１００ｍＭほう酸緩衝液（ｐＨ９．２）で２回洗浄し、同緩衝液液で５ｍｇ乾燥菌体／ｍｌに希釈し、４５℃、４時間、水浴中で処理を行った。
（３）反応
反応液は１２３２ｍＭフマル酸、６１６ｍＭエチレンジアミン、９２４ｍＭの水酸化マグネシウム、２０ｍｇ乾燥菌体／ｍｌの上記処理菌体または同濃度の未処理菌体を含み、６Ｎ水酸化ナトリウムによってｐＨ８．５に調整したものを用い、３０℃、２０日間反応させた。また、６Ｎ水酸化ナトリウムによりｐＨを８．５に保った。
反応液中のＳ，Ｓ−ＥＤＤＳ、Ｌ−リンゴ酸の定量は実施例１記載の方法で行った。
（４）結果
【００７６】
【表７】

【００７７】
【発明の効果】
本発明により、ＥＤＤＳアーゼ活性とフマラーゼ活性を含む微生物についてＥＤＤＳアーゼ活性を低下させずにフマラーゼ活性のみをを選択的に除去することができ、フマル酸損失の少ない光学活性アミノポリカルボン酸製造用微生物触媒を提供し得る。
さらに、アルカリ処理された該微生物またはその処理物は、たとえ該処理によりＥＤＤＳアーゼ活性が部分的に損失していたとしても、相対的にフマラーゼ活性除去割合が大きければ、フマル酸損失の少ない光学活性アミノポリカルボン酸製造用触媒となり得るため、本発明の効果を得ることができる。
【００７８】
【配列表】

【図面の簡単な説明】
【図１】この図は、プラスミドｐＥＤＳ００１の制限酵素地図である。
【図２】この図は、プラスミドｐＥＤＳ００２の制限酵素地図である。このプラスミドは、プラスミドｐＥＤＳ００１を制限酵素ＫｐｎＩ、ＢａｍＨＩで切断して得た約３．９ｋｂの断片をｐＵＣ１８に挿入したものである。
【図３】この図は、プラスミドｐＥＤＳ００３の制限酵素地図である。このプラスミドは、プラスミドｐＥＤＳ００１を制限酵素ＫｐｎＩ、ＢａｍＨＩで切断して得た約２．６ｋｂの断片をｐＵＣ１８に挿入したものである。
【図４】この図は、プラスミドｐＥＤＳ０２０の制限酵素地図である。このプラスミドは、プラスミドｐＥＤＳ００３を制限酵素ＫｐｎＩ、ＢａｍＨＩで切断して得た約２．６ｋｂの断片をｐＵＣ１１９に挿入したものである。
【図５】この図は、プラスミドｐＳＥ００１の構築図である。
【図６】この図は、プラスミドｐＳＪ０３４の構築図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides a method for removing fumarase activity of a microorganism having ethylenediamine-N, N'-disuccinic acid ethylenediamine lyase activity, a microorganism having reduced fumarase activity or a processed product thereof, and a fumarase activity obtained by the method. The present invention relates to a method for producing an optically active aminopolycarboxylic acid from fumaric acid and a compound having an amino group in the presence of a reduced microorganism or a processed product thereof.
[0002]
[Prior art]
Ethylenediamine-N, N'-disuccinic ethylenediamine lyase is used as a catalyst when producing optically active aminopolycarboxylic acid from fumaric acid and a compound having an amino group, and the obtained optically active aminopolycarboxylic acid is a metal such as heavy metal. Since it has a unique property of capturing ions and is susceptible to biodegradation, it is expected to be used as a chelating agent, a detergent builder or a photographic bleach.
[0003]
The present inventors have previously found a novel lyase activity of a microorganism that converts fumaric acid and ethylenediamine to S, S-ethylenediamine-N, N'-disuccinic acid (hereinafter, this lyase is referred to as ethylenediamine-N, N '). -Ethylenediamine succinate lyase, abbreviated as EDDSase), and a method for efficiently producing an optically active aminopolycarboxylic acid from fumaric acid and various amines utilizing the present catalytic action has been proposed [Japanese Patent Application Laid-Open No. 9-1997]. No. 140390, U.S. Pat. No. 5,707,836]. However, these EDDSase-bearing microorganisms combine fumarases that are widely present in the living world and hydrate a substantial portion of the common substrate, fumaric acid, into malic acid. It is difficult to obtain the desired optically active aminopolycarboxylic acid at a high yield.
[0004]
As a method for removing fumarase activity, a method of treating a microorganism having aspartase activity with an acid (see Japanese Patent Publication No. 3-55103), and a method of removing a microorganism belonging to the genus Brevibacterium having the same aspartase activity, Is known under the alkaline conditions in the presence of L-aspartic acid and ammonium ions (see Japanese Patent Publication No. 4-80678).
[0005]
[Problems to be solved by the invention]
However, it was experimentally confirmed that the former method cannot be used for the microorganism of the present invention because the activity loss of EDDSase is larger than that of fumarase. In addition, the latter method is advantageous for the reaction for producing L-aspartic acid which can be subjected to the reaction together with the treatment liquid containing a large amount of L-aspartic acid, but the operability and economical efficiency for other reactions. There was difficulty in terms of.
[0006]
The EDDSase of the present invention is a novel enzyme different from the above-mentioned aspartase, and therefore it was completely unknown how to selectively remove only fumarase activity without losing EDDSase activity.
Therefore, an object of the present invention is to selectively remove only fumarase activity from a microorganism having EDDSase activity and fumarase activity without losing EDDSase activity.
[0007]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, the inventors have found that a microorganism having EDDSase activity or a processed product thereof is impaired by treating the microorganism with an EDDSase activity in an alkaline aqueous solution in the presence of salts. The present inventors have found a method for selectively removing fumarase activity without using the same, and have reached the present invention. That is, in the first aspect, the present invention provides a method for producing a microorganism having ethylenediamine-N, N'-ethylenediamine succinate ethylenediamine lyase activity or a treated product thereof in the presence of salts having a concentration of 5 to 1000 mM and a pH of 8.0 to 10.5. And a method for removing fumarase activity of the microorganism or a processed product thereof, characterized in that the microorganism is treated with an alkaline aqueous solution.
Here, the salts are selected from the group consisting of sodium, potassium, ammonia and salts of amines of boric acid, phosphoric acid, hydrochloric acid, sulfuric acid, acetic acid, oxalic acid, fumaric acid, maleic acid and ethylenediamine-N, N′-disuccinic acid. Can be selected.
[0008]
The microorganism or a processed product thereof is a bacterium consisting of the genus Pseudomonas, the genus Paracoccus, the genus Sphingomonas and the genus Brevundimononas; the genus Escherichia or the genus Rhodococus belonging to the genus Escherichia (Rhodococcus). A transformant in which a gene encoding EDDSase has been introduced into a bacterial host; and a product obtained by treating the bacterium or the transformant. Specifically, they are selected from Pseudomonas sp. TN-131 strain (FERM BP-5418), Paracoccus sp. TNO-5 strain (FERM BP-6546), Sphingomonas sp. TN-28 strain (FERM BP-5419), Brevundimonasp. TN-30 strain (FERM BP-5417) and Brevundimonas sp. TN-3 strain (FERM BP-5886); Escherichia coli JM109 strain (ATCC53323), Rhodococcus rhodochrous ATCC17895 strain and Rodococcus rhodochrous J-1 strain (FERM BP-1478) which transformed the EDDS gene into a host. Selected from the group consisting of a treated product of the bacterium or the transformant.
[0009]
As used herein, the term "processed product" refers to a product obtained by treating a crushed cell, a cell extract, a crude enzyme or a purified enzyme extracted from the cell, an immobilized cell or enzyme, a stabilizing agent, or the like. Cells or enzymes. The present invention also provides, in the second aspect, a microorganism having EDDSase activity or a processed product thereof, characterized in that fumarase activity is reduced. Such a microorganism or a processed product thereof also includes a microorganism obtained by the method of the present invention or a processed product thereof.
[0010]
As used herein, the term "reduced" with respect to the fumarase activity means that the fumarase activity is reduced as compared to the original microorganism or the processed product, or that the fumarase activity is substantially not contained. The present invention further provides, in a third aspect, the use of the above microorganism or a processed product thereof, which has reduced fumarase activity, in the production of an optically active aminopolycarboxylic acid from fumaric acid and a compound having an amino group.
Specifically, the present invention relates to the following general formula [1]
[0011]
Embedded image

[0012]
A method for producing an optically active aminopolycarboxylic acid represented by the formula: wherein fumaric acid and a general formula [2]
[0013]
Embedded image

[0014]
And isolating the optically active aminopolycarboxylic acid from the resulting reaction mixture, wherein in the above formulas [1] and [2] , R₁And R₂May be the same or different, and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, provided that R₁And R₂Are not both hydrogen atoms and the substituents are selected from the group consisting of amino groups, cyano groups, nitro groups, halogens, hydroxyl groups, carboxyl groups and ether groups;₃And R₄Is R₁And R₂Is the same as R₁And R₂Represents a group having a structure in which at least one of the amino groups of the formula (1) is bonded via its nitrogen atom to the carbon atom of the ethylene group of succinic acid.
[0015]
Here, the compound having an amino group is, for example, ethylenediamine, 1,3-propanediamine, 2-methyl-1,3-propanediamine, 2-hydroxy-1,3-propanediamine, 1,4-butanediamine, C2-C6 alkane- or cycloalkane-diamines such as 1,5-pentanediamine, 1,6-hexanediamine, 1,2-cyclohexanediamine, 1,3-cyclohexanediamine, and 1,4-cyclohexanediamine Phenylenediamines such as 1,2-phenylenediamine, 1,3-phenylenediamine, 1,4-phenylenediamine; and glycine, 3-aminopropionic acid, 2-aminopropionic acid, iminodiacetic acid, 3,3′- Selected from the group consisting of monoamines such as iminodipropionic acid and glutamic acid That.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
As described above, the present invention aims to suppress side reactions in the production of an optically active aminopolycarboxylic acid, and to obtain a microbial catalyst from which fumarase activity has been selectively removed while maintaining EDDSase activity by the treatment. The purpose is to: Therefore, the effects of the present invention can be obtained as long as fumaric acid is used as a raw material and the catalyst is applied to the production of an optically active aminopolycarboxylic acid.
Examples of these applications include a case where an optically active aminopolycarboxylic acid represented by the following general formula [1] is produced from a mixture of a compound having an amino group represented by the following general formula [2] and fumaric acid. Can be mentioned:
[0017]
Embedded image

[0018]
Embedded image

[0019]
Where R₁And R₂May be the same or different, and each represents a hydrogen atom (provided that R₁And R₂Are not hydrogen atoms), a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, R₃And R₄Is R₁And R₂Same as or R₁And R₂Represents a group having a structure in which at least one of the amino groups is bonded to the carbon atom of the ethylene group of succinic acid via its nitrogen atom, and the substituent is an amino group, a cyano group, a nitro group, a halogen, a hydroxyl group, Carboxyl group, ether group and the like.
[0020]
Examples of the compound having an amino group represented by the general formula [2] include ethylenediamine, 1,3-propanediamine, 2-methyl-1,3-propanediamine, 2-hydroxy-1,3-propanediamine, Alkanes having 2 to 6 carbon atoms, such as 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, 1,2-cyclohexanediamine, 1,3-cyclohexanediamine, and 1,4-cyclohexanediamine Or phenylenediamines such as cycloalkane-diamines, 1,2-phenylenediamine, 1,3-phenylenediamine, 1,4-phenylenediamine, and glycine, 3-aminopropionic acid, 2-aminopropionic acid, iminodiacetic acid And monoamines such as 3,3'-iminodipropionic acid and glutamic acid Etc. can be mentioned, ethylene diamine is typical.
[0021]
Representative examples of the optically active aminopolycarboxylic acid represented by the general formula [1] obtained in the present invention include (S, S) -form ethylenediamine-N, N'-disuccinic acid, 1,3 -Propanediamine-N, N'-disuccinic acid, 2-methyl-1,3-propanediamine-N, N'-disuccinic acid, 2-hydroxy-1,3-propanediamine-N, N'-disuccinic acid, 1,4-butanediamine-N, N'-disuccinic acid, 1,5-pentanediamine-N, N'-disuccinic acid, 1,6-hexanediamine-N, N'-disuccinic acid, 1,2-cyclohexane Diamine-N, N'-disuccinic acid, 1,3-cyclohexanediamine-N, N'-disuccinic acid, 1,4-cyclohexanediamine-N, N'-disuccinic acid, 1,2-phenylenediaminedisuccinic acid Alkanediamine-N, N'-disuccinic acid, such as 1,3-phenylenediaminedisuccinic acid and 1,4-phenylenediaminedisuccinic acid, cycloalkanediamine-N, N'-disuccinic acid or phenylenediamine-N, N '-Disuccinic acid; and (S) -form aspartic acid-N-monoacetic acid, aspartic acid-N-monopropionic acid, aspartic acid-N-2-propionic acid, aspartic acid-N, N-diacid Examples thereof include acetic acid, aspartic acid-N, N-dipropionic acid, and aspartic acid-N-2-glutaric acid, and (S, S) -ethylenediamine-N, N'-disuccinic acid is typical.
[0022]
The microorganisms to be subjected to the fumarase activity removal treatment of the present invention are as described below. The fumarase activity removal treatment is carried out by treating cells obtained by culturing or their treated products (crushed cells, cell extract, extracted crude / purified enzymes, immobilized cells or enzymes, drug treatment (stabilization treatment, etc.) ) Cells or enzymes). In addition, after the cultivation, it can be performed directly on the cells in the culture solution. Further, another treatment may be added after the fumarase activity removal treatment.
[0023]
The removal treatment of the fumarase activity can be performed by immersing the above-mentioned microbial cells and the like in an aqueous alkaline solution in the presence of salts. The aqueous alkaline solution may be an organic solvent freely miscible with water, such as methanol or ethanol, or a mixture of other organic solvents in a range that is miscible with water.
The salts are not particularly limited, as long as they can be dissolved in the aqueous alkali solution of the present invention. For example, boric acid, phosphoric acid, hydrochloric acid, sulfuric acid, acetic acid, oxalic acid, fumaric acid, maleic acid, salts of sodium, potassium, ammonia, amines and the like such as ethylenediamine-N, N'-disuccinic acid, and amines Preferred are monoamines such as alkanediamine having 2 to 6 carbon atoms such as ethylenediamine, propanediamine, butanediamine, and hexanediamine, and triethanolamine.
[0024]
The salt concentration ranges from 5 to 1000 mM, preferably from 10 to 500 mM. If the amount is less than 5 mM, the effect is not sufficient, and if the amount exceeds 1000 mM, the effect reaches a plateau.
The pH of the alkaline aqueous solution is in the range of 8 to 10.5, preferably 8.5 to 10, and more preferably 9 to 9.5, and is adjusted to a predetermined pH by changing the ratio of the acid and the base. Can be. Above all, the use of boric acid, phosphoric acid or Good's buffer is preferred in terms of both adjustment of salt concentration and pH.
[0025]
The processing temperature is usually in the range of freezing temperature to 55 ° C. The processing time varies depending on various conditions, but is about 1 minute to about 1 month. By setting the processing temperature to a higher temperature, the processing time can be shortened. Further, although the treatment time is extended, it is possible to remove the fumarase activity during refrigerated storage under the above conditions.
These processing operations can be performed in either a batch or continuous manner.
[0026]
The microorganism of the present invention includes any microorganisms having EDDSase and fumarase activities. For example, bacteria belonging to the genus Pseudomonas, the genus Paracoccus, the genus Sphingomonas and the genus Brevundimonas, and the host belonging to the genus Escherichia or the genus Rhodococcus belonging to the genus Escherichia or Rhodococcus to the host. A transformant into which gene DNA encoding EDDSase has been introduced can be mentioned as an example.
[0027]
Specifically, Pseudomonas sp. TN-131 strain (FERM BP-5418), Paracoccus sp. TNO-5 strain (FERM BP-6546), Sphingomonas sp. TN-28 strain (FERM BP-5419), Brevundimonas sp. TN-30 strain (FERMBP-5417) and TN-3 strain (FERM BP-5886), and Escherichia coli JM109 strain (Escherichia coli ATCC53323), Rhodococcus rhodochrous ATCC17895 and RhodococroscoscusFrochuscoscusFroccocrostroscoscusFroccocrostroscus strains. 1478).
[0028]
Among the above microorganisms, TN-131 strain, TNO-5 strain, TN-28 strain, TN-30 strain, and TN-3 strain were newly isolated from nature by the present inventors, and were identified by Deposited at the Institute of Biotechnology and Industrial Technology. The bacteriological properties of these strains are as follows.
[0029]

[0030]

[0031]

[0032]

[0033]
The above mycological properties are described in Bergey's Manual of Systematic Bacteriology Vol. 1 (1984) and Bergey's Manual of Deterministic Bacteriology, 9th edition (1994), strain TN-131 belongs to the genus Pseudomonas, and Bergey's Manual of Systematic Medicine. 1 (1984), the TNO-5 strain belongs to the genus Paracoccus and is classified as Bergey's Manual of Determinative Bacteriology, 9th edition (1994) and Microbiol. Immunol.34TN-28 strain belongs to the genus Sphingomonas, Bergey's Manual of Determinative Bacteriology 9th edition (1994) and Int. J. Syst. Bacteriol.44,When classified according to 499 (1994), the TN-30 strain and the TN-3 strain are identified as bacteria belonging to the genus Brevundimonas. In addition, it has been confirmed that the TN-3 strain is a diminuta strain.
[0034]
Escherichia coli strain JM109 (Escherichia coli ATCC 53323) and Rhodococcus rhodochrous ATCC 17895 are known and can be easily obtained from the American Type Culture Collection (ATCC). Using these strains as a host, transformants into which plasmids pEDS020 and pSE001 containing gene DNA encoding a protein having EDDSase activity of the TN-3 strain were introduced were used. E. coli JM109 / pEDS020 (FERM BP-6161; transferred to an international deposit on November 10, 1997) and Rhodococcus rhodochrous ATCC 17895 / pSE001 (FERM BP-6548; transferred to an international deposit on October 15, 1998), respectively. Deposited at the Institute of Biotechnology and Industrial Technology, Institute of Technology. Hereinafter, a method for producing these transformants will be described.
[0035]
(1) Preparation of TN-3 strain chromosomal DNA
The TN-3 strain was placed in a 100 ml EDDS medium (0.2% ethylenediamine-N, N'-disuccinic acid, 0.2% glucose, 0.1% bacto yeast extract, 0.05% polypeptone, 0.28% sodium sulfate). , 0.1% magnesium sulfate 7H₂O, 2.5% (v / v) 1 M phosphate buffer (pH 7.0), 0.5% (v / v) metal salt mixture solution (composition in 100 ml; magnesium chloride 6H₂O8g, calcium chloride 0.8g, manganese sulfate-4H₂O 0.6g, ferric chloride ・ 6H₂O.12g, zinc sulfate 0.06g)), shake-cultured at 30 ° C for 4 days, and then collected, and the cells were collected in a Saline-EDTA solution (0.1 M EDTA, 15 M NaCl (pH 8.0)). Suspend in 4 ml, add 8 mg of lysozyme, shake at 37 ° C. for 1 hour, and freeze. Next, 10 ml of a Tris-SDS solution (1% SDS, 0.1 M NaCl, 0.1 M Tris (pH 9.0)) was added with gentle shaking, and proteinase K (manufactured by Merck) (final concentration: 1 mg) was further added. Add and shake for 1 hour at 37 ° C. Next, an equal amount of TE-saturated phenol was added, and the mixture was stirred (TE: 10 mM Tris, 1 mM EDTA (pH 8.0)), centrifuged, the upper layer was added, and twice the amount of ethanol was added. Phenol is removed sequentially with 90%, 80% and 70% ethanol. Next, the DNA is dissolved in 3 ml of TE buffer, a ribonuclease A solution (heat treated at 100 ° C. for 15 minutes) is added to a concentration of 10 mg / ml, and shaken at 37 ° C. for 30 minutes. Further, proteinase K is added, and the mixture is shaken at 37 ° C. for 30 minutes. Then, an equal amount of TE-saturated phenol is added, and the mixture is centrifuged to separate the upper and lower layers. After repeating this operation twice for the upper layer, the same amount of chloroform (containing 4% isoamyl alcohol) is added and the same extraction operation is repeated (hereinafter, this operation is referred to as phenol treatment). Then, double the amount of ethanol is added to the upper layer, and the DNA is wound up and collected with a glass rod to obtain a chromosomal DNA sample.
[0036]
(2) Preparation of purified enzyme
The TN-3 strain was cultured in 2 liters of EDDS medium with shaking at 30 ° C. for 4 days, and then collected, suspended in 100 ml of 10 mM sodium phosphate buffer (pH 8.0, containing 1 mM dithiothreitol), and suspended. The cells are disrupted by a sonicator and centrifuged at 12,000 rpm for 20 minutes. Ammonium sulfate is added to the obtained supernatant so as to be 30% saturated, left at 4 ° C. for 30 minutes, and then centrifuged. Ammonium sulfate is added to the obtained supernatant so as to be 60% saturated, left at 4 ° C. for 30 minutes, and then centrifuged. The obtained precipitate is dissolved in 10 ml of 10 mM sodium phosphate buffer (pH 8.0, containing 1 mM dithiothreitol) to obtain a partially purified enzyme solution.
[0037]
This partially purified enzyme solution is further purified by ion exchange chromatography. That is, the partially purified enzyme solution was allowed to flow through a column (φ10 mm × 20 cm) packed with DEAE-Sephacryl (manufactured by Pharmacia) buffered with 10 mM sodium phosphate buffer (pH 8.0) containing 1 mM dithiothreitol to be adsorbed. Was. After washing the column with 40 ml of the same buffer, the enzyme is eluted with a linear gradient of 0 to 0.6 M KCl, and fractionated in 2 ml portions. The fractions in which EDDSase activity is observed are collected and used as a purified enzyme solution. When this fraction was analyzed by SDS-polyacrylamide gel electrophoresis, a substantially uniform single band having a molecular weight of about 60,000 was detected.
[0038]
(3) Analysis of N-terminal amino acid sequence of purified enzyme and amino acid sequence of internal peptide The purified enzyme obtained in step (2) is directly or trypsin-digested and then fractionated by SDS-polyacrylamide gel electrophoresis. Then, electroblotting is performed on a PVDF membrane (Immobilon Psq (manufactured by Millipore)). The blotted membrane is stained with Coomassie Brilliant Blue, and the stained band is cut out and subjected to amino acid sequence analysis. The amino acid sequence is analyzed using a PSQ-1 type amino acid analyzer manufactured by Shimadzu Corporation. The results are shown below.
[0039]
a) N-terminal amino acid sequence of untreated enzyme: (molecular weight: about 60,000);
Xaa-Thr-Pro-His-Asn-Pro-Asp-Ala
(SEQ ID NO: 6; where Xaa indicates Met or deletion)
b) trypsin partially decomposed product: (molecular weight: about 50,000);
Glu-Ile-Gly-Ser-Val-Gly-Lys-Met-Glu-Ile-Gly-Arg-Xaa-Ala-Asn-Asp-Leu-Arg-Asn-Arg
(SEQ ID NO: 7; Xaa represents an unidentified amino acid residue.)
[0040]
c) trypsin partially decomposed product: (molecular weight about 10,000);
Ala-Ser-Gly-Ala-Lys-Ala-Pro-Glu-Phe-Gln-Glu-Leu-Tyr-Asp-Phe-Glu-Ala-Ala-Xaa-Leu-Xaa-Leu
(SEQ ID NO: 8; Xaa represents an unidentified amino acid residue.)
[The molecular weight in parentheses indicates the molecular weight of the fractionated peptide. ]
[0041]
(4) Preparation of probe
Based on the amino acid sequence information obtained in the step (3), synthetic DNAs as shown in SEQ ID NOs: 4 and 5 are prepared and used as primers (primers # 1 and # 2) and obtained in the step (1). Polymerase chain reaction is performed using the chromosomal DNA of the obtained TN-3 strain as a template.
That is, 1 μl of the TN-3 strain chromosomal DNA, 10 μl of a 10-fold concentration reaction buffer, 4 μl of 10 mM dNTP, 1 μl of each of primers # 1 and # 2 (corresponding to 100 pmol concentration), and 1 μl of ExTaq (Takara Shuzo) were added to make 100 μl. This solution is subjected to 30 cycles of incubation at 95 ° C. for 30 seconds (denaturation step), 55 ° C. for 30 seconds (annealing step), 72 ° C. for 2 minutes (extension step). After completion of the reaction, chloroform extraction is performed three times, and the amplified DNA is recovered by ethanol precipitation. After separation by 1.0% agarose electrophoresis, a DNA fragment of about 300 bp which is considered to encode the EDDSase gene of the TN-3 strain is obtained. The DNA fragment thus obtained is labeled using a DIG DNA Labeling kit (manufactured by Boehringer Mannheim) to obtain a probe.
[0042]
(5) Preparation of DNA library
To 10 μl of the chromosomal DNA of the TN-3 strain, 5 μl of a 10-fold concentration restriction enzyme reaction buffer, 33 μl of sterilized water and 2 μl of a restriction enzyme KpnI are added, and the mixture is allowed to react at 37 ° C. for 16 hours. Agarose electrophoresis is performed, and a DNA fragment of 6.5 Kb to 5.5 Kb is excised from the gel and recovered using DNA PREP (Diatron). This DNA fragment is inserted into the KpnI site of the E. coli vector pUC18 using a ligation kit (Takara Shuzo) to prepare a recombinant DNA library.
[0043]
The pUC18 fragment used for ligation is prepared as follows. To 2 μl of the pUC18 stock solution, 5 μl of 10-fold concentration buffer for restriction enzyme, 40 μl of sterilized water, and 3 μl of KpnI restriction enzyme were added, reacted at 37 ° C. for 2 hours, phenol treated, ethanol precipitated, dried and sterilized to 50 μl Dissolve in water. Further, 1 μl of alkaline phosphatase (Takara Shuzo Co., Ltd.), 10 μl of 10-fold concentration buffer and 39 μl of sterilized water are added, and the mixture is reacted at 65 ° C., subjected to phenol treatment, ethanol precipitation, dried and dissolved in sterilized water.
[0044]
(6) Preparation of E. coli transformant and selection of recombinant DNA
Escherichia coli JM109 strain was inoculated into 1 ml of LB medium (1% bactotryptone, 0.5% bactoeast extract, 0.5% NaCl), aerobically precultured at 37 ° C. for 5 hours, and 100 ml of this culture was subjected to SOB Medium (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO₄, 1 mM MgCl₂) Add to 40 ml and incubate at 18 ° C for 20 hours. After collecting the culture by centrifugation, a cold TF solution (20 mM PIPES-KOH (pH 6.0), 200 mM KCl, 10 mM CaCl₂, 40 mM MnCl₂) Is added, and left at 0 ° C. for 10 minutes, and then centrifuged again. After removing the supernatant, the precipitated E. coli was suspended in 3.2 ml of a cold TF solution, 0.22 ml of dimethyl sulfoxide was added, and the mixture was allowed to stand at 0 ° C. for 10 minutes. 10 μl of the solution (DNA library) containing the recombinant plasmid prepared in step (5) was added to 200 μl of the competent cells thus prepared, left at 0 ° C. for 30 minutes, and then subjected to heat shock at 42 ° C. for 30 seconds. After cooling at 2 ° C. for 2 minutes, SOC medium (20 mM glucose, 2% bactotryptone, 0.5% bactoeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO 4)₄, 1 mM MgCl₂) Add 1 ml and incubate with shaking at 37 ° C for 1 hour. This is spread on an LB agar medium containing 100 μg / ml of ampicillin in 200 μl portions, and cultured at 37 ° C. A transformant having an EDDSase gene is selected by a colony hybridization method from a transformant colony grown on an agar medium. That is, a transformant grown on an agar medium was transferred to a nylon membrane (Biodyne A: manufactured by Pall Corporation), and the DNA was fixed by dissolving the cells, followed by the probe ( The colonies having the target recombinant DNA are selected using a DIG Luminescent Detection Kit (manufactured by Boehringer Mannheim).
[0045]
(7) Preparation of recombinant plasmid
The transformant selected in the step (6) is cultured overnight at 37 ° C. in 100 ml of LB medium, collected, washed with sterilized water, and treated with solution I (2 mM glucose, 10 mM EDTA, 25 mM Tris-HCl (pH 8. 5) and 25 mg of lysozyme are added and left at 0 ° C. for 30 minutes. 10 ml of solution II (1N NaOH, 5% SDS) is added and left at 0 ° C. for 5 minutes. 7.5 ml of solution III (3M sodium acetate (pH 4.8)) is added and left at 0 ° C. for 30 minutes. This was centrifuged, 50 ml of ethanol was added to the supernatant, and the supernatant was further removed by centrifugation. 5 ml of solution IV (10 mM sodium acetate, 50 mM Tris-HCl (pH 8.0)) and ribonuclease A solution (10 mg / ml) Add 2.5 μl and leave at room temperature for 20 minutes. To this is added 12 ml of ethanol, the plasmid is recovered by centrifugation, rinsed with 70% ethanol, dried and dissolved in 0.4 ml of sterile water. The recombinant plasmid thus obtained is named pEDS001.
[0046]
(8) Preparation of restriction map and identification of EDDSase gene region
The plasmid pEDS001 obtained in the step (7) is cut with several kinds of restriction enzymes to prepare a restriction map (FIG. 1). In addition, subcloning is performed as usual. That is, pEDS001 was digested with restriction enzymes KpnI and BamHI, and pUC18 was ligated with the same digested with the same restriction enzymes. Escherichia coli JM109 strain was transformed to obtain a plasmid (pEDS002) into which a fragment of about 3.9 Kb was inserted. ) (FIG. 2) and a plasmid (pEDS003) (FIG. 3) into which a fragment of about 2.6 Kb has been inserted. After digesting each plasmid with restriction enzymes BamHI, EcoRI, SacI, SalI, etc., agarose electrophoresis is performed, and a fragment to which the probe hybridizes is identified by Southern hybridization.
[0047]
(9) Determination of base sequence
The nucleotide sequence around the region specified in the step (8) is determined using a Pharmacia fluorescent sequencer ALFII. As a result, the base sequence shown in SEQ ID NO: 1 is obtained, and an open reading frame having the amino acid sequence shown in SEQ ID NO: 2 is found. As a result of comparison with NBRF (National Biomedical Research Foundation), this gene has a homology of 20 to 30% with delta-crystallin or argininosuccinate lyase, and both enzymes have fumaric acid and amine amino acids. ) Is known to have an activity of catalyzing the condensation or decomposition reaction. The nucleotide sequence of this open reading frame is shown in SEQ ID NO: 3.
[0048]
(10) Preparation of plasmid pEDS020 and Escherichia coli transformant, and EDDSase activity of the transformant
To 0032 μl of the recombinant plasmid pEDS containing the EDDSase gene obtained in step (8), 2 μl of a 10-fold concentration restriction enzyme buffer, 15 μl of sterilized water, and 1 μl of a restriction enzyme KpnI are added and reacted at 37 ° C. for 2 hours. . The plasmid is recovered by ethanol precipitation, and after drying, 17 μl of sterilized water, 2 μl of a 10-fold concentration restriction enzyme buffer, and 1 μl of a restriction enzyme BamHI are added and reacted at 37 ° C. for 2 hours. A fragment of about 2.6 Kb is prepared from the reaction solution by agarose electrophoresis and inserted into an E. coli vector pUC119. Using the ligation solution thus prepared, Escherichia coli JM109 is transformed to obtain a desired plasmid. The plasmid prepared here is named pEDS020 (FIG. 4), and the transformant is named JM109 / pEDS020.
[0049]
JM109 / pEDS020 was inoculated into 1 ml of LB medium containing 50 mg / l of ampicillin and cultured with shaking at 37 ° C for 8 hours, and the whole amount was added to 40 ml of LB medium (containing 50 mg / l ampicillin and 1 mM isopropyl-β-thiogalactoside). The cells were inoculated and cultured at 37 ° C. for 30 hours. The obtained culture is washed with 10 mM sodium phosphate buffer (pH 8.0) and suspended in 2 ml of the same buffer. A part of the obtained cell suspension is suspended in 50 ml of a pH 8.0 aqueous solution containing 342 mM fumaric acid and 171 mM ethylenediamine and reacted for 24 hours. After removing the cells from the reaction solution by centrifugation, the produced ethylenediamine-N, N'-disuccinic acid is analyzed using HPLC (WAKOSIL 5C8 (manufactured by Wako Pure Chemical Industries, Ltd.) [eluent: 10 mM tetrahydroxyhydroxide]. n-Butyl ammonium and 0.4 mM CuSO₄50 mM phosphoric acid, pH 2]). As a result, 50 mM of S, S-ethylenediamine-N, N'-disuccinic acid was produced.
[0050]
(11) Construction of plasmid pSE001
To 2 μl of the plasmid pEDS020 prepared in the step (10), 2 μl of a buffer solution for 10-fold concentration restriction enzyme, 15 μl of sterilized water, and 1 μl of restriction enzyme XhoI are added and reacted at 37 ° C. for 2 hours. The plasmid is recovered by ethanol precipitation, and after drying, 15 μl of sterilized water, 2 μl of a 10-fold concentration buffer for klenow fragment, 2 μl of a 10 mM dNTP solution, and 1 μl of klenow fragment are added and reacted at 37 ° C. for 2 hours. The DNA fragment was recovered by ethanol precipitation, dried, then added with 8 μl of sterile water, 1 μl of XbaI linker solution, 16 μl of Ligation kit (Takara Shuzo) solution A and 4 μl of solution B, and reacted at 16 ° C. for 4 hours. After that, it is transformed into JM109. A plasmid is prepared from the obtained transformant to obtain a plasmid in which the XhoI site of pEDS020 has been converted to an XbaI site. To 2 µl of the obtained plasmid, 2 µl of a 10-fold concentration buffer for restriction enzyme, 15 µl of sterilized water, and 1 µl of restriction enzyme EcoRV are added and reacted at 37 ° C for 2 hours. The DNA fragment was recovered by ethanol precipitation, dried, then added with 8 μl of sterile water, 1 μl of Sse8387I linker solution, 16 μl of Ligation kit (Takara Shuzo) solution A and 4 μl of solution B, and reacted at 16 ° C. for 4 hours. And then transformed into JM109. A plasmid is prepared from the obtained transformant to obtain a plasmid (pEDS027) in which the EcoRV site has been converted to an Sse8387I site. To 2 μl of the obtained plasmid, 2 μl of a buffer solution for 10-fold concentration restriction enzyme, 14 μl of sterilized water, 1 μl of restriction enzyme XbaI and 1 μl of Sse8387I are added and reacted at 37 ° C. for 2 hours. Thereafter, the band was separated by 0.7% agarose electrophoresis, and the 1.7 Kb band was recovered and inserted into the XbaI-Sse8387I site of the plasmid pSJ034 having a strong promoter activity for Rhodococcus bacteria to prepare a recombinant plasmid pSE001 ( (Fig. 5). pSJ034 is prepared from plasmid pSJ023 (see Japanese Patent Application No. 9-65618) by the steps shown in FIG. pSJ023 has been deposited as a transformant Rhodococcus rhodochrous ATCC12674 / pSJ023 (FERM BP-6232; transferred to the International Depositary on January 21, 1998) with the National Institute of Advanced Industrial Science and Technology.
[0051]
(12) Transformation of Rhodococcus bacteria and EDDSase activity of the transformant
Cells in the logarithmic growth phase of Rhodococcus rhodochrous ATCC 17895 strain are collected by a centrifugal separator, washed three times with ice-cold sterile water, and suspended in sterile water. 1 μl of the plasmid pSE001 and 10 μl of the cell suspension are mixed and cooled on ice. A suspension of DNA and cells is placed in a cuvette, and subjected to electric pulse processing at 2.0 KV, 200 OHMS by a gene transfer apparatus Gene Pulser (BIO RAD). The electric pulse treatment solution was allowed to stand for 10 minutes under ice-cooling, subjected to heat shock at 37 ° C. for 10 minutes, and then subjected to MYK medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄) Add 500 ml, leave at 30 ° C for 5 hours, apply to 50 mg / l kanamycin-containing MYK agar medium, and culture at 30 ° C for 3 days. The resulting transformant is designated as ATCC 17895 / pSE001.
[0052]
The transformant thus prepared is inoculated into 10 ml of MYK medium (containing 50 mg / l kanamycin) and cultured with shaking at 30 ° C for 72 hours. 1 ml of the medium was mixed with 100 ml of medium (1.5% glucose, 1% sodium glutamate, 0.1% Bacto yeast extract, 0.05% KH₂PO₄, 0.05% K₂HPO₄, 0.05% magnesium sulfate, pH 7.2, containing 50 mg / l kanamycin) and cultured at 30 ° C. for 60 hours. Next, the obtained cells were subjected to a reaction and an analysis according to the method described in step (10). As a result, 36 mM of S, S-ethylenediamine-N, N'-disuccinic acid was produced.
[0053]
The reaction for producing the optically active aminopolycarboxylic acid represented by the formula [1] is carried out in water or a buffer solution (for example, a phosphate buffer, a carbonate buffer or a borate buffer). Strains or processed products thereof (ie, crushed cells, cell extracts, crude or purified enzymes extracted from cells, immobilized cells or enzymes, cells treated with agents such as stabilizers, etc.) Or selected from enzymes) with a mixture of fumaric acid and an amino group-containing compound of formula [2].
[0054]
In general, the reaction can be carried out at a temperature of 0 to 50C, preferably 5 to 35C, and a pH of 5 to 11, preferably 6 to 10. The concentration of fumaric acid and the amino group-containing compound represented by the formula [2] can vary depending on the reaction temperature and pH used, and their concentrations can each range from 0.1% to a saturated concentration. The amount of the microorganism or the processed product thereof is usually 0.01 to 5.0% based on the amount of the substrate. The reaction can be performed either batchwise or continuously.
[0055]
Known techniques such as microbial removal, concentration, chromatography (eg, HPLC), crystallization and the like can be used to isolate the resulting aminopolycarboxylic acid from the reaction mixture at the completion of the reaction.
The specific procedure for producing the aminopolycarboxylic acid can be performed in the same manner as the procedure described in JP-A-9-140390 or the corresponding US Pat. No. 5,707,836. The contents of these patent publications and U.S. patents are incorporated herein by reference.
[0056]
【Example】
Next, the present invention will be specifically described with reference to the following examples.
<Example 1>
(1) Culture
Pseudomonas sp. TN-131 strain, Paracoccus sp. The TNO-5 strain, Sphingomonas sp. TN-28 strain, Brevundimonas sp. One platinum loop of the TN-30 strain and the TN-3 strain was picked from the slant medium, inoculated into the following medium, and cultured at 30 ° C. aerobically with shaking for 4 days.
[0057]
Medium composition (pH 7.5, 100 ml):
0.2 g of ethylenediamine-N, N'-disuccinic acid
0.2 g of glucose
Yeast extract 0.1g
Polypeptone 0.05g
Magnesium sulfate 7H₂O 0.1g
0.28g of sodium sulfate
Phosphate buffer 25 mM
Metal salt mixture solution * 0.5 ml
[* Metal salt mixture solution (100 ml): magnesium chloride 6H₂O 8 g, calcium chloride 0.8 g, manganese sulfate 4H₂O 0.6g, ferric chloride ・ 6H₂O 0.12 g, zinc sulfate 0.06 g]
[0058]
(2) Removal treatment of fumarase activity
The cell culture solution is collected in a centrifuge tube, collected by centrifugation at 10,000 rpm at 5 ° C. for 15 minutes, washed twice with a 50 mM borate buffer (pH 9.2), and dried with the same buffer to obtain 5 mg of a dried bacterial cell. And treated in a water bath at 45 ° C. for 6 hours. Note that sampling was performed after a lapse of 2 hours.
[0059]
(3) Activity measurement of EDDSase and fumarase
The cells subjected to the above-described fumarase removal treatment were subjected to EDDSase and fumarase activity measurements, and the residual activity was determined based on the activity of the untreated cells as 100%.
The activity of EDDSase was measured using a reaction solution containing 684 mM fumaric acid, 342 mM ethylenediamine, and 342 mM magnesium hydroxide and adjusted to pH 8.5 with 6N NaOH at a cell concentration of 1/10 of the treatment solution at 30 ° C. overnight. The (S, S) -ethylenediamine-N, N'-disuccinic acid (S, S-EDDS) produced by the reaction was quantified by the following operation.
[0060]
The activity of fumarase was measured using a reaction solution containing 68.4 mM L-sodium malate and 50 mM borate buffer and adjusted to pH 8.5 with 6N NaOH at a cell concentration of 1/10 of the treatment solution at 30 ° C. The reaction was allowed to proceed overnight, and the fumaric acid produced was quantified by the following procedure.
[0061]
Quantification of reaction products:
Analysis of S, S-EDDS, fumaric acid and L-malic acid was performed by liquid chromatography after removing insolubles in the reaction solution by centrifugation at 15,000 rpm at 5 ° C. for 5 minutes. As a quantitative column, WAKOSIL 5C8 (Wako Pure Chemical Industries, Ltd.) [eluent: 10 mM tetra-n-butylammonium hydroxide and 0.4 mM CuSO₄Containing 50 mM phosphoric acid, pH 2], and MCI GEL CRS 10W (manufactured by Mitsubishi Chemical Corporation) [eluent: 10 mM CuSO₄〕It was used.
(4) Result
[0062]
[Table 1]

[0063]
<Example 2>
(1) Culture of transformant
Escherichia coli JM109 / pEDS020
Take one platinum loop from the slant medium, inoculate into an LB medium (1% bactotryptone, 0.5% bactoeast extract, 0.5% NaCl) containing 50 mg / l ampicillin, and shake for 8 hours at 37 ° C. Cultured. This was inoculated into an LB medium (containing 50 mg / l ampicillin, 1 mM isopropyl-b-galactoside) in an amount of 2.5%, and cultured at 37 ° C for 30 hours under aerobic shaking.
[0064]
Rhodococcus rhodochrous ATCC 17895 / pSE001
Take one platinum loop from the slant medium and add 50 mg / l kanamycin to MYK medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄), And cultured with shaking at 30 ° C. for 72 hours. This was added to a GGY medium (1.5% glucose, 1% sodium glutamate, 0.1% Bacto yeast extract, 0.05% K₂HPO₄, 0.05% KH₂PO₄, 0.05% magnesium sulfate, 50 mg / l kanamycin, pH 7.2) and inoculated at 30 ° C for 60 hours with aerobic shaking.
[0065]
(2) Removal treatment of fumarase activity
Same as in the first embodiment.
(3) Activity measurement of EDDSase and fumarase
Same as in the first embodiment.
(4) Result
[0066]
[Table 2]

[0067]
<Example 3>
(1) Culture of transformant
Escherichia coli JM109 / pEDS020 was cultured in the same manner as in Example 2.
(2) Glutaraldehyde treatment and fumarase activity removal treatment
The bacterial cell culture was collected in a centrifuge tube, collected by centrifugation at 10,000 rpm at 5 ° C. for 15 minutes, and washed twice with 50 mM borate buffer (pH 9.2). The cells were treated at 4 ° C. for 2 hours at a final concentration of 5 mg dry cells / ml in the same buffer containing glutaraldehyde at a final concentration of 1 mM, and then treated with 100 mM borate buffer (pH 9.2). The cells were washed twice and diluted with the same buffer to 5 mg dry cells / ml. The glutaraldehyde-treated cells and untreated cells diluted to the same concentration were subjected to a fumarase activity removal treatment in a water bath at 45 ° C. for 6 hours.
(3) Activity measurement of EDDSase and fumarase
Same as in the first embodiment.
(4) Result
[0068]
[Table 3]

[0069]
<Example 4>
(1) Culture of transformant
Same as Example 3.
(2) Removal treatment of fumarase activity
The bacterial cell culture solution is collected in a centrifuge tube, collected by centrifugation at 10,000 rpm, 5 ° C., and 15 minutes, and then diluted with 50 mM borate buffer adjusted to the pH shown in Table 4 with 6N NaOH or 5N sulfuric acid. After washing twice, each buffer was diluted to 5 mg dry cells / ml and treated in a water bath at 45 ° C. for 2 hours.
(3) Activity measurement of EDDSase and fumarase
Same as in the first embodiment.
(4) Result
[0070]
[Table 4]

[0071]
<Example 5>
(1) Culture of transformant
Same as Example 3.
(2) Removal treatment of fumarase activity
The bacterial cell culture is collected in a centrifuge tube, and collected by centrifugation at 10,000 rpm at 5 ° C. for 15 minutes. Then, the salt solution described in Table 5 (pH is adjusted to 9.2 with 6N NaOH or 5N sulfuric acid, respectively). ), Diluted with each salt solution to 5 mg dry cells / ml, and treated in a water bath at 45 ° C for 2 hours.
(3) Activity measurement of EDDSase and fumarase
Same as in the first embodiment.
(4) Result
[0072]
[Table 5]

[0073]
<Example 6>
(1) Culture of transformant
Same as Example 3.
(2) Removal treatment of fumarase activity
The bacterial cell culture solution is collected in a centrifuge tube, and collected by centrifugation at 10,000 rpm, 5 ° C., and 15 minutes, washed twice with 100 mM borate buffer (pH 9.2), and dried with the same buffer to obtain 5 mg of dry bacteria. And treated in a water bath at the temperature and time shown in Table 6.
(3) Activity measurement of EDDSase and fumarase
Same as in the first embodiment.
(4) Result
[0074]
[Table 6]

[0075]
<Example 7>
(1) Culture of transformant
Same as Example 3.
2) Fumarase activity removal treatment
The cell culture solution is collected in a centrifuge tube, collected by centrifugation at 10,000 rpm at 5 ° C. for 15 minutes, washed twice with 100 mM borate buffer (pH 9.2), and dried at 5 mg with the same buffer. The cells were diluted to cells / ml and treated in a water bath at 45 ° C. for 4 hours.
(3) Reaction
The reaction solution contains 1232 mM fumaric acid, 616 mM ethylenediamine, 924 mM magnesium hydroxide, 20 mg dry cells / ml of the above treated cells or the same concentration of untreated cells, and adjusted to pH 8.5 with 6N sodium hydroxide. The reaction was carried out at 30 ° C. for 20 days. The pH was kept at 8.5 with 6N sodium hydroxide.
S, S-EDDS and L-malic acid in the reaction solution were quantified by the method described in Example 1.
(4) Result
[0076]
[Table 7]

[0077]
【The invention's effect】
Advantageous Effects of Invention According to the present invention, a microorganism for producing an optically active aminopolycarboxylic acid, which can selectively remove only a fumalase activity without lowering the EDDSase activity from a microorganism containing an EDDSase activity and a fumalase activity, and has a small loss of fumaric acid A catalyst may be provided.
Furthermore, even if the microorganism or the processed product thereof is alkali-treated, even if the EDDSase activity is partially lost by the treatment, the optical activity with less fumaric acid loss is relatively high if the removal rate of the fumalase activity is relatively large. Since it can be a catalyst for producing aminopolycarboxylic acid, the effects of the present invention can be obtained.
[0078]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a restriction map of plasmid pEDS001.
FIG. 2 is a restriction map of plasmid pEDS002. This plasmid is obtained by cutting a plasmid pEDS001 with restriction enzymes KpnI and BamHI and inserting a fragment of about 3.9 kb into pUC18.
FIG. 3 is a restriction map of plasmid pEDS003. This plasmid is obtained by cutting a plasmid pEDS001 with restriction enzymes KpnI and BamHI, and inserting a fragment of about 2.6 kb into pUC18.
FIG. 4 is a restriction map of plasmid pEDS020. This plasmid is obtained by cutting a plasmid pEDS003 with restriction enzymes KpnI and BamHI and inserting a fragment of about 2.6 kb into pUC119.
FIG. 5 is a diagram showing the construction of plasmid pSE001.
FIG. 6 is a diagram showing the construction of plasmid pSJ034.

Claims (14)

Treating a microorganism having ethylenediamine-N, N'-disuccinate ethylenediamine lyase (EDDSase) activity or a treated product thereof with an alkaline aqueous solution having a pH of 8.0 to 10.5 in the presence of salts having a concentration of 5 to 1000 mM. A method for removing fumarase activity of the microorganism or a processed product thereof. The salts are selected from the group consisting of sodium, potassium, ammonia and amine salts of boric acid, phosphoric acid, hydrochloric acid, sulfuric acid, acetic acid, oxalic acid, fumaric acid, maleic acid, and ethylenediamine-N, N'-disuccinic acid. The method according to claim 1, wherein the salt is at least one salt. The microorganism or a processed product thereof is a bacterium consisting of Pseudomonas, Paracoccus, Sphingomonas and Brevndimonas; The method according to claim 1 or 2, wherein the method is selected from the group consisting of a transformant obtained by introducing a gene encoding EDDSase into a bacterial host; and a processed product of the bacterium or the transformant. The microorganism or a processed product thereof is obtained from Pseudomonas sp. TN-131 strain (FERM BP-5418), Paracoccus sp. TNO-5 strain (FERM BP-6546), Sphingomonas sp. TN-28 strain (FERM BP-5419), Brevundimonas sp. TN-30 strain (FERM BP-5417) and Brevundimonas sp. TN-3 strain (FERM BP-5886); Escherichia coli JM109 strain (ATCC53323), Rhodococcus rhodochrous ATCC17895 strain and Rodococcus rhodochrous J-1 strain (FERM BP-1478) which transformed the EDDS gene into a host. The method according to claim 3, wherein the method is selected from the group consisting of: a body; and a processed product of the bacterium or the transformant. The processed product is a crushed cell, a cell extract, a crude enzyme or a purified enzyme extracted from the cell, an immobilized cell or enzyme, a cell consisting of a cell or enzyme treated with a stabilizer. 5. The method according to claim 1, wherein the method is selected. The method according to any one of claims 1 to 5, wherein the salt concentration is in the range of 10 mM to 500 mM. The method according to any one of claims 1 to 6, wherein the pH of the aqueous alkaline solution is 8.5 to 10. The method according to any one of claims 1 to 6, wherein the pH of the alkaline aqueous solution is 9 to 9.5. The method according to any one of claims 1 to 8, wherein the temperature of the treatment ranges from a freezing temperature to 55 ° C. The method according to any one of claims 1 to 9, the time of the treatment, characterized in that it is a period of about 1 minute to about 1 month. A microorganism having EDDSase activity and having reduced fumarase activity, or a processed product thereof, which is obtained by removing the framase activity by the method according to any one of claims 1 to 10, and comprising a pseudomonas ( Bacteria consisting of the genera Pseudomonas, Paracoccus, Sphingomonas and Brevundimonas; bacteria belonging to the genus Escherichia or Rhodococcus genus belonging to the genus Escherichia coli belonging to the genus Escherichia coli belonging to the genus Escherichia coli belonging to the genus Rhodococcus to the genus Escherichia or Rhodococcus. The microorganism or a processed product thereof, which is selected from the group consisting of an introduced transformant; and a processed product of the bacterium or the transformed product . Pseudomonas sp. TN-131 strain (FERM BP-5418), Paracoccus sp. TNO-5 strain (FERM BP-6546) ), Sphingomonas sp. TN-28 strain (FERM BP-5419), Brevundimonas sp. TN-30 strain (FERM BP-5417) and Brevundimonas sp. TN-3 strain (FERM BP-5886); Escherichia coli JM109 strain (ATCC53323), Rhodococcus rhodochrous ATCC17895 strain and Rodococcus rhodochrous J-1 strain (FERM BP-1478) which transformed the EDDS gene into a host. The microorganism according to claim 11, which is selected from the group consisting of a body; and a processed product of the bacterium or the transformant. The microorganism or a processed product thereof according to claim 11 or 12, wherein the ratio of the residual activity of the EDDSase activity to the fumarase activity is 0.03 to 0.27. A reaction between fumaric acid and ethylenediamine in the presence of a catalyst comprising the microorganism according to any one of claims 11 to 13 or a processed product thereof, and S, S-ethylenediamine- forming from the resulting reaction mixture. N, N '- disuccinic comprises isolating the acid, S, S- ethylenediamine -N, N' - manufacturing method of disuccinic acid.

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